Although crystallographic studies have provided a detailed description of the conserved core structure of the HIV-1 surface glycoprotein (gp120), much less is known about the structure and function of the first two hyper-variable regions (VIV2), which protrude from the core surface and often undergo extensive insertions and deletions over the course of disease. Elimination of glycosylation sites or deletion of amino acid residues from the VIV2 domain can influence a number of molecular interactions that are critical to virus entry. However, such studies may not fully describe the natural functions of V1V2, and few studies have comprehensively examined the consequences of natural variability within the V1V2 region during virus transmission or disease progression. Here we will define the structure-function relationships of naturally occurring V1V2 regions as they pertain to heterosexual transmission and virus evolution in Env molecules derived from a large heterosexual couple cohort in Zambia. Our preliminary studies demonstrated that a homogeneous virus population with compact length and restricted N-linked glycosylation in VIV2 were found in the recipient partners of eight heterosexual transmission pairs; however, the viral quasispecies of each donor partner contained up to nine different VIV2 variants, ranging as much as 3-fold in size and 9-fold in the number of glycosylation sites. These data suggest that the molecular determinants of heterosexual transmission are related to the structure of V1V2, and V1V2-chimeric Envs will be constructed from five !transmission pairs and utilized to define this relationship. To better understand the extensive variation that emerges in VIV2 following the acute infection period, the course of sequence evolution in VIV2 will be followed in longitudinal samples collected over a minimum of three years from ten newly identified seroconvertors in the same cohort. We will construct V1V2-chimeric Env molecules to investigate the ,biological consequences of temporal changes within the VIV2 region during infection. Through these studies, we will test the hypotheses that viruses with compact V1V2 regions establish infection and that progressive increases in length and glycosylation arise over the course of infection that contribute to immune evasion and replication fitness, but interfere with virus transmission.